Microfluidic Impedance Cytometry Research Articles

Optimizing Microfluidic Impedance Cytometry by Bypass Electrode Layout Design.

Microfluidic impedance cytometry (MIC) has emerged as a popular technique for single-cell analysis. Traditional MIC electrode designs consist of a pair of (or three) working electrodes, and their detection performance needs further improvements for microorganisms. In this study, we designed an 8-electrode MIC device in which the center pair was defined as the working electrode, and the connection status of bypass electrodes could be changed. This allowed us to compare the performance of layouts with no bypasses and those with floating or grounding electrodes by simulation and experiment. The results of detecting Φ 5 μm beads revealed that both the grounding and the floating electrode outperformed the no bypass electrode, and the grounding electrode demonstrated the best signal-to-noise ratio (SNR), coefficient of variation (CV), and detection sensitivity. Furthermore, the effects of different bypass grounding areas (numbers of grounding electrodes) were investigated. Finally, particles passing at high horizontal positions can be detected, and Φ 1 μm beads can be measured in a wide channel (150 μm) using a fully grounding electrode, with the sensitivity of bead volume detection reaching 0.00097%. This provides a general MIC electrode optimization technology for detecting smaller particles, even macromolecular proteins, viruses, and exosomes in the future.

On the compatibility of single-cell microcarriers (nanovials) with microfluidic impedance cytometry.

We investigate for the first time the compatibility of nanovials with microfluidic impedance cytometry (MIC). Nanovials are suspendable crescent-shaped single-cell microcarriers that enable specific cell adhesion, the creation of compartments for undisturbed cell growth and secretion, as well as protection against wall shear stress. MIC is a label-free single-cell technique that characterizes flowing cells based on their electrical fingerprints and it is especially targeted to cells that are naturally in suspension. Combining nanovial technology with MIC is intriguing as it would represent a robust framework for the electrical analysis of single adherent cells at high throughput. Here, as a proof-of-concept, we report the MIC analysis of mesenchymal stromal cells loaded in nanovials. The electrical analysis is supported by numerical simulations and validated by means of optical analysis. We demonstrate that the electrical diameter can discriminate among free cells, empty nanovials, cell-loaded nanovials, and clusters, thus grounding the foundation for the use of nanovials in MIC. Furthermore, we investigate the potentiality of MIC to assess the electrical phenotype of cells loaded in nanovials and we draw directions for future studies.

Microfluidic impedance cytometry with flat-end cylindrical electrodes for accurate and fast analysis of marine microalgae.

Marine microalgae play an increasingly significant role in addressing the issues of environmental monitoring and disease treatment, making the analysis of marine microalgae at the single-cell level an essential technique. For this, we put forward accurate and fast microfluidic impedance cytometry to analyze microalgal cells by assembling two cylindrical electrodes and microchannels to form a three-dimensional detection zone. Firstly, we established a mathematical model of microalgal cell detection based on Maxwell's mixture theory and numerically investigated the effects of the electrode gap, microalgal positions, and ion concentrations of the solution on detection to optimize detection conditions. Secondly, 80 μm stainless steel wires were used to construct flat-ended cylindrical electrodes and were then inserted into two collinear channels fabricated using standard photolithography techniques to form a spatially uniform electric field to promote the detection throughput and sensitivity. Thirdly, based on the validation of this method, we measured the impedance of living Euglena and Haematococcus pluvialis to study parametric influences, including ion concentration, cell density and electrode gap. The throughput of this method was also investigated, which reached 1800 cells per s in the detection of Haematococcus pluvialis. Fourthly, we analyzed live and dead Euglena to prove the ability of this method to detect the physiological status of cells and obtained impedances of 124.3 Ω and 31.0 Ω with proportions of 15.9% and 84.1%, respectively. Finally, this method was engineered for the analysis of marine microalgae, measuring living Euglena with an impedance of 159.61 Ω accounting for 3.9%, dead Euglena with an impedance of 36.43 Ω accounting for 10.1% and Oocystis sp. with an impedance of 55.00 Ω accounting for about 81.0%. This method could provide a reliable tool to analyze marine microalgae for monitoring the marine environment and treatment of diseases owing to its outstanding advantages of low cost, high throughput and high corrosion resistance.

A microfluidic impedance cytometry device for robust identification of H. pluvialis.

H. pluvialis contains rich oleic acid and astaxanthin, which have important applications in the fields of biodiesel and biomedicine. Detection of live H. pluvialis is the prerequisite to obtaining oleic acid and astaxanthin. For this purpose, we successfully developed a reliable microfluidic impedance cytometry for the identification of live H. pluvialis. Firstly, we established a simulation model for detecting H. pluvialis based on their morphology and studied the effect of medium conductivity on the impedance of H. pluvialis at different frequencies. From the simulations, we determined that the optimal solution conductivity for the detection of H. pluvialis was 1500 μS cm-1 and studied the frequency responses of the impedance of H. pluvialis. Secondly, we fabricated the microchannels and stainless-steel detection electrodes and assembled them into microfluidic impedance cytometry. The frequency dependence of live and dead H. pluvialis was explored under different frequencies, and live and dead H. pluvialis were distinguished at a frequency of 1 MHz. The impedance of live H. pluvialis at the frequency of 1 MHz ranges from 33.73 to 52.23 Ω, while that of dead ones ranges from 13.05 to 19.59 Ω. Based on these findings, we accomplished the identification and counting of live H. pluvialis in the live and dead sample solutions. Furthermore, we accomplished the identification and counting of live H. pluvialis in the mixed samples containing Euglena and H. pluvialis. This approach possesses the promising capacity to serve as a robust tool in the identification of target microalgae, addressing a challenge in the fields of biodiesel and biomedicine.

Real-time impedance-activated dielectrophoretic actuation for reconfigurable manipulation of single flowing particles.

This work presents an innovative all-electrical platform for selective single-particle manipulation. The platform combines microfluidic impedance cytometry for label-free particle characterization and dielectrophoresis for contactless multi-way particle separation. The microfluidic chip has a straightforward coplanar electrode layout and no particle pre-focusing mechanism is required. An original online algorithm analyzes the impedance signals of each incoming particle and regulates in real time the dielectrophoretic voltages according to a desired control logic. As a proof-of-concept, three operation modes are demonstrated on a mixture of 8, 10, and 12 μm diameter beads: (i) particle position swapping across the channel axis, irrespective of particle size, (ii) size-based particle separation, irrespective of particle position, and (iii) sorting of a selected sequence of particles. As a perspective, the versatility of impedance cytometry and dielectrophoresis, and the possibility of configuring alternative control logics, hold promise for advanced particle and cell manipulation.

Low-cost sheath-less microfluidic impedance cytometry for point-of-care applications

Low-cost sheath-less microfluidic impedance cytometry for point-of-care applications

Rapid Assessment of Susceptibility of Bacteria and Erythrocytes to Antimicrobial Peptides by Single-Cell Impedance Cytometry.

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight antibiotic-resistant infections. In most cases, they kill bacteria by making their membrane permeable and therefore exhibit low propensity to induce bacterial resistance. In addition, they are often selective, killing bacteria at concentrations lower than those at which they are toxic to the host. However, clinical applications of AMPs are hindered by a limited understanding of their interactions with bacteria and human cells. Standard susceptibility testing methods are based on the analysis of the growth of a bacterial population and therefore require several hours. Moreover, different assays are required to assess the toxicity to host cells. In this work, we propose the use of microfluidic impedance cytometry to explore the action of AMPs on both bacteria and host cells in a rapid manner and with single-cell resolution. Impedance measurements are particularly well-suited to detect the effects of AMPs on bacteria, due to the fact that the mechanism of action involves perturbation of the permeability of cell membranes. We show that the electrical signatures of Bacillus megaterium cells and human red blood cells (RBCs) reflect the action of a representative antimicrobial peptide, DNS-PMAP23. In particular, the impedance phase at high frequency (e.g., 11 or 20 MHz) is a reliable label-free metric for monitoring DNS-PMAP23 bactericidal activity and toxicity to RBCs. The impedance-based characterization is validated by comparison with standard antibacterial activity assays and absorbance-based hemolytic activity assays. Furthermore, we demonstrate the applicability of the technique to a mixed sample of B. megaterium cells and RBCs, which paves the way to study AMP selectivity for bacterial versus eukaryotic cells in the presence of both cell types.

Development of a Novel Design of Microfluidic Impedance Cytometry for Improved Sensitivity and Cell Identification.

A long-standing issue for microfluidic impedance cytometry devices is the accuracy in determining the size of cells during counting and measurements. In this paper, we introduce a novel design that produces a homogeneous electric field in the sensing region and demonstrates higher accuracy than traditional designs in cell counting and sizing, reducing the reliance on cell focusing and signal postprocessing. The concept is validated, and the increased accuracy of the device over traditional designs is demonstrated through the use of finite element simulations to generate suitable data sets for particle trajectories and model expected signal variations.

Label-Free Sensing of Cell Viability Using a Low-Cost Impedance Cytometry Device.

Cell viability is an essential physiological status for drug screening. While cell staining is a conventional cell viability analysis method, dye staining is usually cytotoxic. Alternatively, impedance cytometry provides a straightforward and label-free sensing approach for the assessment of cell viability. A key element of impedance cytometry is its sensing electrodes. Most state-of-the-art electrodes are made of expensive metals, microfabricated by lithography, with a typical size of ten microns. In this work, we proposed a low-cost microfluidic impedance cytometry device with 100-micron wide indium tin oxide (ITO) electrodes to achieve a comparable performance to the 10-micron wide Au electrodes. The effectiveness was experimentally verified as 7 μm beads can be distinguished from 10 μm beads. To the best of our knowledge, this is the lowest geometry ratio of the target to the sensing unit in the impedance cytometry technology. Furthermore, a cell viability test was performed on MCF-7 cells. The proposed double differential impedance cytometry device has successfully differentiated the living and dead MCF-7 cells with a throughput of ~1000 cells/s. The label-free and low-cost, high-throughput impedance cytometry could benefit drug screening, fundamental biological research and other biomedical applications.

A Simple Micromilled Microfluidic Impedance Cytometer with Vertical Parallel Electrodes for Cell Viability Analysis.

Microfluidic impedance cytometry has been demonstrated as an effective platform for single cell analysis, taking advantage of microfabricated features and dielectric cell sensing methods. In this study, we present a simple microfluidic device to improve the sensitivity, accuracy, and throughput of single suspension cell viability analysis using vertical sidewall electrodes fabricated by a widely accessible negative manufacturing method. A microchannel milled through a 75 µm platinum wire, which was embedded into poly-methyl-methacrylate (PMMA), created a pair of parallel vertical sidewall platinum electrodes. Jurkat cells were interrogated in a custom low-conductivity buffer (1.2 ± 0.04 mS/cm) to reduce current leakage and increase device sensitivity. Confirmed by live/dead staining and electron microscopy, a single optimum excitation frequency of 2 MHz was identified at which live and dead cells were discriminated based on the disruption in the cell membrane associated with cell death. At this frequency, live cells were found to exhibit changes in the impedance phase with no appreciable change in magnitude, while dead cells displayed the opposite behavior. Correlated with video microscopy, a computational algorithm was created that could identify cell detection events and determine cell viability status by application of a mathematical correlation method.

Single-cell impedance cytometry of anticancer drug-treated tumor cells exhibiting mitotic arrest state to apoptosis using low-cost silver-PDMS microelectrodes.

Chemotherapeutic drugs such as paclitaxel and vinblastine interact with microtubules and thus induce complex cell states of mitosis arrest at the G2/M phase followed by apoptosis dependent on drug exposure time and concentration. Microfluidic impedance cytometry (MIC), as a label-free and high-throughput technology for single-cell analysis, has been applied for viability assay of cancer cells post drug exposure at fixed time and dosage, yet verification of this technique for varied tumor cell states after anticancer drug treatment remains a challenge. Here we present a novel MIC device and for the first time perform impedance cytometry on carcinoma cells exhibiting progressive states of G2/M arrest followed by apoptosis related to drug concentration and exposure time, after treatments with paclitaxel and vinblastine, respectively. Our results from impedance cytometry reveal increased amplitude and negative phase shift at low frequency as well as higher opacity for HeLa cells under G2/M mitotic arrest compared to untreated cells. The cells under apoptosis, on the other hand, exhibit opposite changes in these electrical parameters. Therefore, the impedance features differentiate the HeLa cells under progressive states post anticancer drug treatment. We also demonstrate that vinblastine poses a more potent drug effect than paclitaxel especially at low concentrations. Our device is fabricated using a unique sacrificial layer-free soft lithography process as compared to the existing MIC device, which gives rise to readily aligned parallel microelectrodes made of silver-PDMS embedded in PDMS channel sidewalls with one molding step. Our results uncover the potential of the MIC device, with a fairly simple and low-cost fabrication process, for cellular state screening in anticancer drug therapy.

Label-free multidimensional bacterial characterization with an ultrawide detectable concentration range by microfluidic impedance cytometry.

Rapid and accurate identification of bacteria is of great importance to public health in various fields, including medical diagnostics, food safety, and environmental monitoring. However, most existing bacterial detection methods have very narrow detectable concentration ranges and limited detection information, which easily leads to wrong diagnosis and treatment. This work presents a novel high-throughput microfluidic electrical impedance-based multidimensional single-bacterium profiling system for ultrawide concentration range detection and accurate differentiation of viability and Gram types of bacteria. The electrical impedance-based microfluidic cytometry is capable of multi-frequency impedance quantification, which allows profiling of the bacteria size, concentration, and membrane impedance as an indicator of bacterial viability and Gram properties in a single flow-through interrogation. It has been demonstrated that this novel impedance cytometry has an ultrawide bacterial counting range (102-108 cells per mL), and exhibits a rapid and accurate discrimination of viability and Gram types of bacteria in a label-free manner. Escherichia coli (E. coli) has been used as an analog species for the accuracy assessment of the electrical impedance-based bacterial detection system in an authentic complex beverage matrix within 24 hours. The impedance-based quantifications of viable bacteria are consistent with those obtained by the classical bacterial colony counting method (R2 = 0.996). This work could pave the way for providing a novel microfluidic cytometry system for rapid and multidimensional bacterial detection in diverse areas.

An adaptive three-dimensional hydrodynamic focusing microfluidic impedance flow cytometer.

Microfluidic impedance cytometry is emerging as a label-free, low-cost and portable solution for cell analysis. Impedance-based cell or particle characterization is provided by microfluidic and electronic devices. We report the design and characterization of a miniaturized flow cytometer based on a 3-dimensional (3D) hydrodynamic focusing mechanism. A sheath adaptively concentrated the sample laterally and vertically at the bottom of the microchannel, reducing the variance of particle translocation height and increasing the signal-to-noise ratio of the particle impedance pulse. Through simulation and confocal microscopy experiments, it has been verified that an increase in the ratio of sheath to sample decreased the cross-sectional area of the concentrated stream, which can be reduced to 26.50% of the pre-focusing value. The appropriate sheath flow settings increased the impedance pulse amplitude for different particles, and the coefficient of variation reduced by at least 35.85%, contributing to a more accurate representation of the particle impedance characteristic distribution. The system displayed the difference of HepG2 cell impedance before and after drug treatment, which is consistent with the results of flow cytometry, providing a convenient and inexpensive solution for monitoring cell status.

A Simple 3-D Microelectrode Fabrication Process and Its Application in Microfluidic Impedance Cytometry

Microfluidic impedance cytometry (MIC) has wide applications in single-cell analysis. However, the complex microelectrode fabrication process seriously restricts the miniaturization and commercialization of MIC devices. Common microelectrode configurations such as a coplanar electrode, a symmetric electrode, and a liquid electrode have difficulties in meeting the requirements of high detection accuracy and simple fabrication at the same time. Hence, a simple 3-D symmetric microelectrode fabrication process is developed in this article. We design a microchip containing both a microfluidic channel and a microelectrode channel. The alloy with high surface tension and low melting point is used to fabricate the 3-D microelectrodes by injecting the melted alloy into the microelectrode channel with a properly controlled driving pressure. The melted alloy flows into the microelectrode channel and automatically stops at the end of the microelectrode channel due to the effect of surface tension. This process intrinsically provides a high-precision alignment for the 3-D microelectrodes. The fabricated MIC chip is experimentally tested, and the results show that it can effectively count and classify microspheres of different sizes. This study can facilitate the development and commercialization of MIC devices.

A new approach for accurate determination of particle sizes in microfluidic impedance cytometry

In microfluidic impedance cytometry, the change in impedance is recorded as an individual cell passes through a channel between electrodes deposited on its walls, and the particle size is inferred from the amplitude of the impedance signal using calibration. However, because the current density is nonuniform between electrodes of finite width, there could be an error in the particle size measurement because of uncertainty about the location of the particle in the channel cross section. Here, a correlation is developed relating the particle size to the signal amplitude and the velocity of the particle through the channel. The latter is inferred from the time interval between the two extrema in the impedance curve as the particle passes through a channel with cross-sectional dimensions of 50 μm (width) × 30 μm (height) with two pairs of parallel facing electrodes. The change in impedance is predicted using 3D COMSOL finite-element simulations, and a theoretical correlation that is independent of particle size is formulated to correct the particle diameter for variations in the cross-sectional location. With this correlation, the standard deviation in the experimental data is reduced by a factor of two to close to the standard deviation reported in the manufacturer specifications.

Label-Free Microfluidic Impedance Cytometry for Acrosome Integrity Assessment of Boar Spermatozoa.

Microfluidics and lab-on-chip technologies have been used in a wide range of biomedical applications. They are known as versatile, rapid, and low-cost alternatives for expensive equipment and time-intensive processing. The veterinary industry and human fertility clinics could greatly benefit from label-free and standardized methods for semen analysis. We developed a tool to determine the acrosome integrity of spermatozoa using microfluidic impedance cytometry. Spermatozoa from boars were treated with the calcium ionophore A23187 to induce acrosome reaction. The magnitude, phase and opacity of individual treated and non-treated (control) spermatozoa were analyzed and compared to conventional staining for acrosome integrity. The results show that the opacity at 19 MHz over 0.5 MHz is associated with acrosome integrity with a cut-off threshold at 0.86 (sensitivity 98%, specificity 97%). In short, we have demonstrated that acrosome integrity can be determined using opacity, illustrating that microfluidic impedance cytometers have the potential to become a versatile and efficient alternative in semen analysis and for fertility treatments in the veterinary industry and human fertility clinics.

Enhancing signals of microfluidic impedance cytometry through optimization of microelectrode array.

Microfluidic impedance cytometry shows a great value in biomedical diagnosis. However, the crosstalk between neighboring microelectrodes strongly weakens the impedance signal. Hereby, we demonstrate a novel microfluidic impedance cytometer consisted of sensing electrodes and ground electrodes (GNDs). The simulation reveals a signal enhancement by more than five times with GNDs compared to that without ones. We also found that the linear correlation between the impedance at a high frequency and that at a low frequency varies as microparticle size changes, which can be used for microparticle classification. The study can help with microelectrode optimization and signal processing for microfluidic impedance analysis.

Deciphering impedance cytometry signals with neural networks.

Microfluidic impedance cytometry is a label-free technique for high-throughput single-cell analysis. Multi-frequency impedance measurements provide data that allows full characterisation of cells, linking electrical phenotype to individual biophysical properties. To efficiently extract the information embedded in the electrical signals, potentially in real-time, tailored signal processing is needed. Artificial intelligence approaches provide a promising new direction. Here we demonstrate the ability of neural networks to decipher impedance cytometry signals in two challenging scenarios: (i) to determine the intrinsic dielectric properties of single cells directly from raw impedance data streams, (ii) to capture single-cell signals that are hidden in the measured signals of coincident cells. The accuracy of the results and the high processing speed (fractions of ms per cell) demonstrate that neural networks can have an important role in impedance-based single-cell analysis.

A New Approach for Accurate Determination of Particle Sizes in Microfluidic Impedance Cytometry

A New Approach for Accurate Determination of Particle Sizes in Microfluidic Impedance Cytometry

Time-domain signal averaging to improve microparticles detection and enumeration accuracy in a microfluidic impedance cytometer.

Microfluidic impedance cytometry is a powerful system to measure micro and nano-sized particles and is routinely used in point-of-care disease diagnostics and other biomedical applications. However, small objects near a sensor's detection limit are plagued with relatively significant background noise and are difficult to identify for every case. While many data processing techniques can be utilized to reduce noise and improve signal quality, frequently they are still inadequate to push sensor detection limits. Here, we report the first demonstration of a novel signal averaging algorithm effective in noise reduction of microfluidic impedance cytometry data, improving enumeration accuracy, and reducing detection limits. Our device uses a 22 µm tall × 100 µm wide (with 30 µm wide focused aperture) microchannel and gold coplanar microelectrodes that generate an electric field, recording bipolar pulses from polystyrene microparticles flowing through the channel. In addition to outlining a modified moving signal averaging technique theoretically and with a model data set, we also performed a compendium of characterization experiments including variations in flow rate, input voltage, and particle size. Multivariate metrics from each experiment are compared including signal amplitude, pulse width, background noise, and signal-to-noise ratio (SNR). Incorporating our technique resulted in improved SNR and counting accuracy across all experiments conducted, and the limit of detection improved from 5 to 1 µm particles without modifying microchannel dimensions. Succeeding this, we envision implementing our modified moving average technique to develop next-generation microfluidic impedance cytometry devices with an expanded dynamic range and improved enumeration accuracy. This can be exceedingly useful for many biomedical applications, such as infectious disease diagnostics where devices may enumerate larger-scale immune cells alongside sub-micron bacterium in the same sample.